Ectoine and Hydroxyectoine Stabilize Antibodies in Spray-Dried
Formulations at Elevated Temperature and during a Freeze/Thaw
Process Purnendu K. Nayak, Meghan Goode, Debby P. Chang, and Karthikan Rajagopal*
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Supporting Information ABSTRACT: Maintenance of protein stability during manufac- ture, storage, and delivery is necessary for the successful development of a drug product. Herein, the utility of two compatible solutesectoine and hydroxyectoinein stabilizing a model protein labeled Fab2 has been investigated. Specifically, the performance of ectoine and hydroxyectoine in stabilizing Fab2 in a spray-dried formulation at elevated temperature and after multiple freeze/thaw cycles has been compared with the performance of a formulation containing trehalose and a formulation containing no excipient as controls. In the solid state at 90 and 37 °C and in freeze concentrate systems, ectoine and hydroxyectoine suppress protein aggregation. Like trehalose, hydroxyectoine also limits N- terminal pyroglutamate formation in Fab2 in the solid state. The extent of protein stabilization is dependent on the excipient concentration in the formulation, but at a 1:1 excipient to protein mass ratio, hydroxyectoine is better than trehalose in stabilizing
Fab2. The results presented here suggest that ectoine and hydroxyectoine are effective excipients for stabilizing therapeutic antibodies.
KEYWORDS: protein stability, excipients, spray drying, solid state, freeze/thaw
■INTRODUCTION Protein-based therapeutics such as antibodies and fragment antibodies are susceptible to physical and chemical degradation reactions that cause a loss in product quality.1−3 The physicochemical stresses encountered during drug product manufacture such as long-term exposure to a certain pH and temperature, interfacial forces at the air−water interface, visible and UV light radiation, and mechanical shear can potentially degrade protein-based therapeutics. Protein degradation in a drug product before it is administered to patients can have unfavorable consequences. For example, high-molecular-weight protein aggregation products may give rise to immunogenicity and/or cause loss in efficacy in vivo.4,5 Design of a robust formulation for maintaining protein stability during drug product manufacture, storage, and delivery is, therefore, necessary for the successful development of protein-based therapeutics.
Excipients are necessary for maintaining protein stability in liquid- and solid-state formulations. Sugars such as sucrose or trehalose suppress protein aggregation in a drug substance in the freeze concentrate and during long-term shelf-life storage as a lyophilized drug product.6,7 The molecular-level mechanisms for protein stabilization by sugars in the freeze concentrate and in the solid state are fundamentally different but the same sugar usually stabilizes the protein in the two states.8,9 In the aqueous phase and in freeze concentrate, excipients enhance protein stability by decreasing the free energy of the native folded state relative to the unfolded state.
Thermodynamic incompatibility between the excipient and protein surface in the aqueous phase induces preferential hydration of the protein surface to stabilize the protein’s native state. In the solid state, however, sugars preferentially interact with the protein surface and substitute for the water/protein interaction in the aqueous phase to stabilize the protein.
Alternatively, sugars can also form a rigid and glassy matrix.
Here, long-range molecule diffusion and short-range structural relaxations that are needed for degradation are suppressed when proteins are immobilized within a rigid matrix.
Consequently, the melting or unfolding temperature (Tm) of a protein in the solid state is significantly higher than in the aqueous phase.
Nature inspires the identification of excipients for stabilizing proteins. Extremophilic microorganisms produce compatible solutes for self-preservation during periods of adverse environ- Received:
April 13, 2020 Revised:
July 11, 2020 Accepted:
July 16, 2020 Published: July 16, 2020 Article pubs.acs.org/molecularpharmaceutics
© 2020 American Chemical Society 3291 https://dx.doi.org/10.1021/acs.molpharmaceut.0c00395
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See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. mental stresses such as heat, osmotic pressure, or extreme desiccation.10−13 For example, trehalose production is upregulated in prokaryotes during periods of extreme desiccation and osmotic stress.14 Compatible solutes are water soluble and uncharged or zwitterionic small molecules and can be classified as sugars (trehalose, sucrose), polyhydroxy alcohols (glycerol, sorbitol, and mannitol), betaines (glycine betaine), and amino acids (proline, glutamine, and alanine) or amino acid derivatives (ectoines).
These solutes are produced intracellularly and have evolved to preserve the protein structure and function, membranes, and cells during adverse environmental conditions. Importantly, many compatible solutes have been investigated as excipients for the stabilization of cells and biomolecules.
Of particular interest here is the utility of ectoine and hydroxyectoine as excipients for stabilizing antibodies. Ectoine and hydroxyectoine are heterocyclic amino acids (Figure 1).
While L-aspartate is biosynthetically converted to ectoine, stereo-specific enzymatic hydroxylation converts ectoine to hydroxyectoine. Stabilization of cells and biomolecules against various physicochemical stresses by ectoines has been reported before.15,16 Ectoines prevent DNA damage because of ionizing radiation17,18 and improve the thermodynamic stability of proteins in the aqueous phase by preferential hydration.19
Hydroxyectoine has been shown to protect lactate dehydro- genase against metal-catalyzed and hydrogen peroxide-induced oxidation20 and improve the thermodynamic stability of
RNase.21 Despite their pharmaceutically relevant protein- stabilization properties, ectoines have not been evaluated for formulating therapeutic antibodies.
The objective of this study is to investigate ectoine and hydroxyectoine as excipients for stabilizing a model protein (labeled Fab2) in a spray-dried formulation at elevated temperature and in the liquid state during a freeze/thaw (F/
T) process. Stability of proteins in the spray-dried form at elevated temperature is necessary in the preparation of polymer rods for sustained release applications. Fab2, a fragment antibody, was selected as a model protein because analytical methods for quantifying aggregation and specific chemical-degradation reactions such as aspartic acid isomer- ization and N-terminal pyroglutamate formation were readily available. In addition, the generality of the concept of protein stabilization by ectoines was also tested by investigating the stability of a model monoclonal antibody (labeled Mab2) in the solid state at 90 °C.
Trehalose stabilization of Fab2 in spray-dried formulations at elevated temperatures has been extensively investigated and reported previously.22 Herein, the performance of ectoine and hydroxyectoine in stabilizing Fab2 has been compared with that of trehalose and with a formulation devoid of an excipient.
Specifically, aggregation and chemical degradation in Fab2 have been investigated after exposing spray-dried formulations to 90 °C for a few hours and to 37 °C for a few weeks. A temperature of 90 °C was selected to assess the suitability of spray-dried formulations for high-temperature processing such as hot-melt extrusion used in the preparation of polymer rods.
A stability study at 37 °C for a few weeks is a meaningful measure of long-term drug product stability under refrigerated conditions (2−8 °C). Furthermore, stability at 37 °C mimics the in vivo temperature and captures the effect of exposing the protein to physiological temperature during its long-term residence within a sustained delivery system. In addition, F/T stability of Fab2 was also studied as a function of all excipients.
Multiple freeze and thaw cycles subject the antibody to similar stresses encountered during drug substance storage. The freezing stress causes the antibody to freeze concentrate, which could present undesired product quality changes.
■MATERIALS AND METHODS Materials. Fab2 and Mab2 were obtained from Genentech (South San Francisco, CA). α-α-Trehalose dihydrate was obtained from Ferro Pfanstiehl Laboratories (Cleveland, OH), and hydroxyectoine and ectoine were obtained from Sigma- Aldrich (St. Louis, MO). Histidine base and histidine−HCl were obtained from Sigma-Aldrich (St. Louis, MO), and polysorbate 20 (PS20) and polysorbate 80 (PS80) were obtained from Spectrum Chemical (New Brunswick, NJ).
Spray Drying of Fab2. Fab2 at 25 mg/mL was dialyzed [molecular weight cutoff(MWCO) = 10,000 Da] against 10 mM histidine/histidine−HCl buffer (pH 5.5) containing
0.01% PS20. The dialysis buffer was changed four times in
16 h, Fab2 was diluted to 10 mg/mL with 10 mM histidine/ histidine−HCl buffer (pH 5.5) containing 0.01% PS20. UV spectroscopy was used to measure the Fab2 concentration (ε =
1.39 mL·cm−1·mg−1 at 280 nm). The required amounts of trehalose dihydrate, ectoine, and hydroxyectoine were added to
Fab2 resulting in solutions with the same excipient/protein
Figure 1. Chemical structure and molecular weight. (A) Trehalose, (B) ectoine, and (C) hydroxyectoine.
Table 1. Summary of Spray-Dried Fab2 Properties E/P excipient in formulation spray-dried particle size (μm) glass transition (Tg) (°C) (mg/mg) (mol/mol)
% monomer (SEC) % main peak (IEC) trehalose 5.2 73
1 137 99.7 87.27 ectoine 7.9 69.4 1 330 99.6 86.82 hydroxyectoine
4.3 72.6 1 297 99.7 87.14 no excipient 9.2 NA 0 0 99.7
87.23 Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
Article https://dx.doi.org/10.1021/acs.molpharmaceut.0c00395
Mol. Pharmaceutics 2020, 17, 3291−3297 3292 (E/P) mass ratio of 1 (see Table 1). The aqueous formulations were spray dried using a Buchi model B290 Mini spray dryer (New Castle, NJ) at an inlet and outlet temperature of 110 ± 2 and 60 ± 2 °C, respectively. The pump power was 8%, and the aspirator was operated at 100% capacity. The liquid feed rate was 3.4 mL/min, and the compressed air flow rate was 600 L/ h. The collected spray-dried powders were transferred to a clean, dry glass vial, closed with a screw cap and stored under nitrogen in a vacuum chamber until further use. All spray-dried powders were further collected into 15 cc Lyo glass vials and secondary dried in a benchtop lyophilizer (Advantage Pro
Lyophilizer, SP Scientific, UK) to further reduce the moisture level.
Spray Drying of Mab2. Mab2 at 30 mg/mL was dialyzed (MWCO = 10,000 Da) against 10 mM sodium citrate buffer (pH 6.5). The dialysis buffer was changed three times in 24 h, and Mab2 was diluted to 10 mg/mL with 10 mM sodium citrate buffer (pH 6.5). 7% (w/v) PS80 was added to each solution to make a final PS80 concentration of 0.07%. UV spectroscopy was used to measure the Mab2 concentration (ε
= 1.66 mL·cm−1·mg−1 at 280 nm). The required amounts of trehalose dihydrate, ectoine, and hydroxyectoine were added to the Mab2 solution to achieve a 1:1 E/P mass ratio, and the solutions were filtered through a 0.22 μm Millex sterile filter.
The aqueous formulations were spray dried using a Buchi model B191 Mini spray dryer (New Castle, NJ) at an inlet and outlet temperature of 90 ± 2 and 60 ± 2 °C, respectively. The pump power was 8%, and the aspirator was operated at 100% capacity. The liquid feed rate was 3.4 mL/min, and the compressed air flow rate was 600 L/h. The collected spray- dried powders were transferred to a clean, dry glass vial, closed with a screw cap and stored under nitrogen in a vacuum chamber until further use. All spray-dried powders were further collected into 15 cc Lyo glass vials and secondary dried in a benchtop lyophilizer (Advantage Pro Lyophilizer, SP Scientific,
UK) to further reduce the moisture level.
Scanning Electron Microscopy. The morphology of the spray-dried particles was imaged with a Quanta 3D (Hillsboro,
OR) FEG scanning electron microscope. The samples were fixed on aluminum stubs with carbon adhesive tape and sputter coated with gold/palladium (Cressington Sputter Coater, TED
Pella, Inc.) to improve their electrical conductivity. Scanning electron microscopy (SEM) images were collected at low voltage to minimize any potential sample damage or surface charging.
Particle Size Characterization by Laser Diffractions.
The particle size distributions of spray-dried Fab2 powders were measured using a Partica LA-950V2 laser diffraction particle size distribution analyzer (Horiba Ltd., Kyoto, Japan).
Approximately 1 mg of spray-dried powder was dispersed in 1 mL of isopropanol, and the dispersion was added dropwise to
50 mL isopropanol until a target light obscuration level was achieved. The refractive index of isopropanol (1.3776) was used to calculate the size distribution using the particle sizing program.
Stability Study at 90 °C for Fab2 and Mab2. Spray- dried powder (2−7 mg, equivalent to 1−1.5 mg Fab2 or Mab2 mass) from each formulation was weighed into 7 mL glass vials. The vials were uncapped and placed in a Binder forced- air convection oven (Bohemia, NY), preheated, and equili- brated to the desired temperature at 90 °C. Samples were removed at 1, 2, 3, 4, and 5 h, capped immediately, and allowed to cool to room temperature. The vial contents were dissolved in purified water such that the final protein concentration was ∼1 mg/mL. The reconstituted aqueous samples were observed for clarity, and visibly clear samples were used for size exclusion chromatography (SEC) and ion- exchange chromatography (IEC) analysis.
Stability Study at 37 °C. Spray-dried powder (2−7 mg, equivalent to 1−1.5 mg Fab2 or Mab2 mass) from each formulation was weighed into 15 mL Lyo tubing type-1 borosilicate glass vials. The vials were stoppered and equilibrated to the desired temperature at 37 °C in a humidity-controlled incubator chamber at 26% relative humidity. Samples were removed at 1, 2, 3, and 4 weeks, and allowed to cool to room temperature. The vial contents were dissolved in purified water such that the final protein concentration was ∼1 mg/mL. The reconstituted aqueous samples were observed for clarity, and visibly clear samples were used for SEC and IEC analysis.
Size Exclusion Chromatography. For Fab2 SEC, high- performance liquid chromatography (HPLC) was performed using an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with a TOSOH TSKgel G2000SWXL (7.8 × 300 mm) column. Samples were analyzed at 25 °C in isocratic mode with 0.20 M K3PO4 and 0.25 M KCl, pH 6.2, as the mobile phase at a flow rate of 0.7 mL/min. A 20 μL sample at
1 mg/mL concentration was injected, and the total run time was 20 min. Absorbance at 280 nm was used for detection.
For Mab2, SEC separation was performed using a TOSOH
TSKgel G3000SWXL (7.8 × 300 mm) column. Samples were analyzed at 25 °C in isocratic mode with 0.20 M K3PO4, 0.25
M KCl, pH 6.2, as the mobile phase at a flow rate of 0.5 mL/ min. A 100 μL sample at 0.5 mg/mL concentration was injected. The total run time was 30 min and absorbance at 280 nm was used for detection.
The SEC peaks were divided into monomers, high- molecular-weight species, and fragments. The percent peak area was calculated by dividing the peak area of each group at each time point by the total peak area.
Ion-Exchange Chromatography. IEC HPLC was performed using an Agilent 1200 series HPLC system (Santa
Clara, CA) on two Dionex (Sunnyvale, CA) ProPac SAX-10 (2
× 250 mm) strong anion-exchange columns connected in series and equipped with a diode array detector. Mobile phase
A (solvent A) was 20 mM Tris buffer, pH 8.2, and mobile phase B (solvent B) was 250 mM sodium chloride dissolved in solvent A. For spray-dried powders, 20 μL of the reconstituted sample in water at 1 mg/mL was injected directly. A linear gradient starting from 100% solvent A at 0 min to 20% solvent
A at 45 min was employed to separate Fab2 charge variants in a total of ∼60 min run time. Absorbance at 280 nm was used for detection. The IEC peaks were separated into a main peak, an acidic peak, and a basic peak.
Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) was performed on a TA Q200 (New Castle
DE) under the modulation conditions. Approximately 2 ± 1 mg sample was weighed in an aluminum pan and hermetically sealed. The sample was equilibrated at 5 °C for 10 min and then heated at 2 °C/min with ±1.00 °C/min modulation to
120 °C and cooled back to 5 °C at 2 °C/min. After the sample was equilibrated again at 5 °C, the same heating and modulation ramp was repeated for a second time all the way to 180 °C. The first heating ramp was done to eliminate any thermal history associated with the sample because of storage or handling conditions. For reporting purposes, the second
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Mol. Pharmaceutics 2020, 17, 3291−3297 3293 heating ramp of the sample was used, which is free from any thermal history. Glass-transition temperature was measured as the midpoint of the sharp transition in the thermogram obtained in reversible mode (Figure S1).
F/T Stability. F/T of Fab2 formulations was done at a 25 mg/mL protein concentration, 1 mL aliquot, and in 2 cc lyophilization vials. For freezing, the samples were kept at −20
°C for 3 h. This was followed by thawing at ambient temperature for 1 h. The F/T cycle was repeated three times for all four formulations in triplicate. The extent of Fab2 aggregation after each F/T cycle was measured using SEC.
■RESULTS Four formulations of Fab2 (at 10 mg/mL) were evaluated for stability after the spray-drying process. All four formulations used pH 5.5 His−HCl/His buffer and 0.01% PS20 (Table 1).
One of the formulations served as a control and had no excipients. The other three formulations contained trehalose or ectoine or hydroxyectoine at a 1:1 excipient to protein mass ratio (E/P). The mole ratio of excipient to protein, however, was different because of the differences in the excipient molecular weight.
All four formulations after spray drying yielded micron-sized particles (Figure S2) with a similar particle morphology (Figure 2). The spray-dried powders were reconstituted in water and analyzed for aggregation using SEC and for chemical degradation using IEC. The monomer content obtained by
SEC and the major charge-variant peak observed by IEC were similar for all formulations after spray drying and comparable to Fab2 before spray drying (Table 1). The size of the spray- dried particle is slightly larger for the formulation devoid of an excipient and the formulation with ectoine as the excipient but no impact on the monomer content or chemical degradation was observed.
Fab2 stability in spray-dried formulations was tested at 90
°C for 5 h and at 37 °C for 4 weeks (Figure 3). Aggregation was maximal in the formulation devoid of an excipient at both temperatures. In the formulation devoid of any excipient, the monomer loss after incubation was 3.8 and 0.6% after 5 h at 90
°C and four weeks at 37 °C, respectively. In formulations containing trehalose, ectoine, and hydroxyectoine, the monomer loss after incubation at 90 °C for 5 h was 1.2, 2.7, and 0.2%, respectively. In formulations containing trehalose, ectoine, and hydroxyectoine, the monomer loss after incubation at 37 °C for four weeks was 0.3, 0.13, and <0.1%, respectively. As expected, a short-term exposure to 90 °C caused more aggregation than a long-term exposure to 37 °C.
Importantly, the excipient-dependent aggregation of Fab2 was consistent at both temperatures, that is, hydroxyectoine provided the maximum protection against aggregation.
The IEC method used an anion exchange column for separating the different charge variant species in the sample.
The peaks to the left and right of the main peak, therefore, correspond to basic and acidic charge variants, respectively.
The characterization and identity of peaks on IEC due to Fab2 degradation has been reported previously.22 Briefly, the peak immediately to the left of the main peak corresponds to pyroglutamate formation that is formed via the intramolecular cyclization of N-terminal glutamic acid and the peaks around
18 and 20 min correspond to the succinimide intermediate product of two and one aspartic acid residues in the sequence, respectively. Formation of a pyroglutamate and a succinimide intermediate product removes a net negative charge and gives rise to basic peaks on anionic IEC.
Exposure of Fab2 to 90 and 37 °C caused chemical degradation but in slightly different ways (Figure 4A,B). The succinimide product formed via the cyclization of a one aspartic acid side chain in Fab2 and the pyroglutamate product (PyroE) formed via the cyclization of N-terminal glutamic acid gave rise to basic peaks around 20 and 23.5 min, respectively, on IEC. Exposing Fab2 to 90 °C caused an increase in both these degradation reactions for all formulations (Figure 4B).
Whereas at 37 °C, the peak around 20 min disappeared and a new peak around 18 min originated in all formulations after stress (Figure 4A). This suggests that a new succinimide product is formed from a second aspartic acid after exposure to
37 °C.
Interestingly, at 90 and 37 °C, the presence of the excipient in the formulation did not influence succinimide formation but the nature of the excipient clearly influenced the extent of pyroglutamate formation. Pyroglutamate formation was quantified by measuring the ratio of the main peak and pyroglutamate peak areas (Figure 4C,D). Importantly, hydroxyectoine and trehalose significantly suppressed pyroglu- tamate formation relative to ectoine- and excipient-free formulations at both temperatures. In formulations containing trehalose or hydroxyectoine, pyroglutamate formation was
Figure 2. Spray-dried particle morphology. SEM micrograph of spray-dried Fab2 without any excipient or with trehalose or ectoine or hydroxyectoine. Scale bar is 5 μm.
Figure 3. Fab2 aggregation in spray-dried formulations. The change in the monomer content as a function of time for Fab2 in spray-dried formulations after exposure to 37 °C for 4 weeks (A) and 90 °C for 5 h (B).
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Mol. Pharmaceutics 2020, 17, 3291−3297 3294 three- to four-fold less relative to formulations containing ectoine or no excipient. At 90 °C, pyroglutamate formation is higher in the formulation containing ectoine than in the formulation without the excipient.
The effect of these excipients in stabilizing a monoclonal antibody (Mab2) was also investigated at 90 °C (Figure S3).
In the absence of any excipient, Mab2 aggregated to 4.5% during the spray-drying process and when the spray-dried formulation was exposed to 90 °C, Mab2 aggregation increased to 10% in 5 h. In the presence of trehalose or ectoine or hydroxyectoine, aggregation was suppressed during the spray- drying process but when spray-dried Mab2 was exposed to 90
°C, the trehalose-containing formulation showed maximal aggregation, followed by hydroxyectoine and ectoine for- mulations. Importantly, relative to the formulation without any excipient, trehalose or ectoine or hydroxyectoine significantly reduced Mab2 aggregation in the spray-dried formulation at 90
°C.
Multiple F/T cycling was used to assess the effect of excipients on protein stability in the freeze concentrate. F/T cycling was conducted by repeating the warming and cooling of the formulation to ambient temperature and −20 °C, respectively. In the absence of any excipient, Fab2 showed a tendency to aggregate with increasing F/T cycles; after three cycles, the monomer content decreased by 0.4% compared to the control. However, the change in the monomer content in the formulations containing trehalose or ectoine or hydrox- yectoine was within assay variability and relatively small compared to the sample without any excipient (Figure 5) suggesting that all the excipients tested perform similarly in protecting Fab2 during F/T stress.
■DISCUSSION Stability in spray-dried formulations was of particular interest because of its utility in sustained drug-delivery systems.
Micron-sized protein particles such as spray-dried powders are preferred for the preparation of hydrophobic and polymeric drug-delivery materials such as polymer rods and polymer− solvent depots.22−24 The spray-dried particle morphology is known to depend on operating conditions and formulation variables. The dimpled nature of the particle surface morphology (Figure 1) is not of any particular concern, but the micrometer size of the spray-dried particles and retention of protein stability after spray drying are critical parameters for drug-delivery applications. Even though spray drying occurs on millisecond timescale, the process exposes the proteins to adverse stresses such as high temperature, shear, and interfacial forces at the air−water interface. Interestingly, the absence or the nature of excipient in the formulation did not influence the morphology of the spray-dried particle and Fab2 stability during the spray-drying process. All formulations, however, contained 0.01% PS20 as the surfactant, which is necessary for limiting protein instability at the air−water interface.
The results presented here suggest that ectoine and hydroxyectoine can function as excipients for stabilizing antibodies in the solid state and during drug substance storage.
Like trehalose, ectoine and hydroxyectoine limit protein aggregation in the solid state at 90 and 37 °C and in the freeze concentrate. In addition, hydroxyectoine can also suppress pyroglutamate formation in the solid-state formula- tions. Based on the data presented here, the performance of ectoine and hydroxyectoine as excipients cannot be directly compared with that of trehalose because the optimal quantity of an excipient required for achieving maximal protein stability could be dependent on the type of excipient. However, at an excipient to protein mass ratio of 1.0, hydroxyectoine is superior to trehalose in limiting protein aggregation in the solid state at 90 and 37 °C.
Direct excipient-protein interaction presumably stabilizes the protein in the solid state. The thermodynamically stabilizing excipient-protein interaction in the solid state replaces hydrogen-bonding interactions between water and protein in the aqueous phase. Compatible solutes have naturally evolved to foster preferential hydration (via solute exclusion) in the aqueous phase and interact favorably with the protein surface in the solid state. Preferential excipient and protein interaction in the solid state implies that maximal stability will be achieved when the excipient completely covers the protein surface. For trehalose, it was demonstrated previously that stability against aggregation is minimized when the trehalose to protein mass ratio (E/P) is 1; increasing trehalose beyond E/P of 1.0 has a
Figure 4. Chemical degradation of Fab2 in spray-dried formulations.
Ion-exchange chromatogram of Fab2 in different formulations after 4 weeks at 37 °C (A) and 5 h at 90 °C (B). Pyroglutamate formation, measured as the ratio of PyroE and main peak areas, with time at 37 (C) and 90 °C (D).
Figure 5. F/T stability. The change in the Fab2 monomer content after multiple F/T cycles for all four formulations.
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Mol. Pharmaceutics 2020, 17, 3291−3297 3295 minimal impact on protein aggregation.22 Determination of optimal quantity of ectoine and hydroxyectoine for maximal protein stability will require the assessment of concentration- dependent stability. Nevertheless, ectoines are effective protein stabilizers at an excipient to protein mass ratio of 1:1.
The data presented here also support the fact that solid-state stability is improved when the excipient forms a rigid and glassy matrix. The superior protein-stabilizing property of hydroxyectoine in the solid state, particularly at elevated temperatures, can be attributed to its superior glass-forming properties.25 Tanne et al. demonstrated that hydroxyectoine is a better glass former than ectoine and affords superior desiccation protection of biological structures. While the symmetric structure of ectoine enables its crystallization, the less-symmetric structure of hydroxyectoine frustrates crystal- lization and forms an amorphous glassy state. In addition, the presence of a hydroxyl group in hydroxyectoine facilitates intermolecular hydrogen-bonding interactions in the amor- phous state. Consequently, the glass transition temperature (Tg) of free hydroxyectoine (Tg = 87 °C) is higher than that of free ectoine (Tg = 47 °C). Perhaps for this reason, ectoine is converted to hydroxyectoine by microorganisms for maintain- ing stability during heat stress and extreme desiccation.25 Even though the incubation temperature is above the Tg of the excipients, no visible change in the spray-dried particles was observed after 5 h at 90 °C for all formulations. For trehalose- containing formulations, no phase change was observed even after heat treatment up to 135 °C.22
It is interesting to find that excipients can also impact the chemical degradation of proteins in the solid state. Chemical degradation in proteins is related to the spontaneous chemical reaction of amino acid side chains such as asparagine deamidation, aspartic acid isomerization, methionine and tryptophan oxidation, and N-terminal glutamic acid pyroglu- tamate formation. Such intramolecular reactions are governed by short-range structural relaxations (β-relaxations), which are primarily rotation around single bonds, and dependent on the protonation state of the functional groups. The pH of a protein in solution before drying is known to dictate its protonation state in the solid state.26 Even though β-relaxations are largely suppressed in the solid state relative to the liquid state and proteins are generally more stable in the solid state, β- relaxations do persist and chemical degradation can be significant provided the exposure temperature is high and the duration is long. The increase in pyroglutamation after 90 °C stress in the ectoine formulation relative to the formulation devoid of an excipient is intriguing. The mechanism requires further investigation but may be related to the stress temperature (90 °C) being much higher than the Tg (47
°C) for free ectoine.
The suppression of pyroglutamate formation in trehalose and hydroxyectoine formulations in the solid state but not in the ectoine formulation and in the formulation devoid of an excipient is interesting. The structural difference between ectoine and hydroxyectoine is a single hydroxyl group and trehalose is a polyhydroxy molecule. That trehalose and hydroxyectoine, but not ectoine, suppress pyroglutamate formation in the solid state possibly implicates hydrogen- bonding interaction between the N-terminal amine and hydroxyl group in the excipient. The amine−hydroxyl interaction via hydrogen bonding in the solid state presumably reduces the rate of pyroglutamate formation in the case of trehalose and hydroxyectoine formulations.
The freezing and thawing process subjects the antibody to stresses typically encountered during drug substance storage.
Ice crystal formation during the freezing process, desiccation of the protein surface in the frozen state, and an increase in the local protein concentration can potentially lead to destabiliza- tion (increase in free energy) and cause protein aggregation.
Protein aggregation rates are expected to be slower because of lower temperature in the freeze concentrate, but long-term storage could cause monomer loss. Product quality differences were observed after F/T stress between the formulations that contained the excipient and the formulation devoid of an excipient. Thus, ectoine and hydroxyectoine are similar to trehalose in that they are kosmotropic solutes, and stabilize proteins by strengthening protein/water hydrogen-bonding interactions under frozen storage conditions. The preferential hydration of the protein surface induced by the excipient improves its conformational stability and eliminates unfolding- driven aggregation. Trout et al. noted that an improvement in conformational stability can sometimes cause colloidal instability at a high protein concentration, which may lead to the formation of reversible aggregates.27 Whether ectoines induce colloidal instability or not is unknown and the effect will also be dependent on the protein sequence. In the case of
Fab2, no such destabilization was observed as a result of F/T stress.
■CONCLUSIONS The stability of a fragment antibody (Fab2) was investigated in spray-dried formulations and after F/T stress as a function of three excipientstrehalose, ectoine, and hydroxyectoine.
Measurement of Fab aggregation in the solid state at 90 °C after a few hours and at 37 °C after a few weeks suggests that all the three excipients limit protein aggregation relative to the formulation devoid of an excipient. Like trehalose, hydrox- yectoine also suppresses pyroglutamate formation in solid-state formulations. Even though excipient stabilization of proteins in solid formulations is dependent on the E/P ratio that is specific to a particular excipient, hydroxyectoine is superior to trehalose and ectoine in limiting aggregation at an excipient to protein ratio of 1.0. The three excipients perform equally well in limiting Fab2 aggregation during F/T stress. In summary, ectoine and hydroxyectoine as excipients can stabilize antibodies in the solid state at elevated temperatures and during F/T stress.
■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharma- ceut.0c00395.
Particle size distribution, DSC thermograms, and Mab2 stability at 90 °C (PDF)
■AUTHOR INFORMATION Corresponding Author Karthikan Rajagopal −Drug Delivery Department, Genentech
Inc., South San Francisco, California 94080, United States; orcid.org/0000-0002-9398-4672; Phone: 001-650-467- 7326; Email: rajagopal.karthikan@gene.com; Fax: 001-650- 225-2764
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Mol. Pharmaceutics 2020, 17, 3291−3297 3296 Authors
Purnendu K. Nayak −Eurofins Lancaster Laboratories,
Lancaster, Pennsylvania 17605, United States Meghan Goode −Drug Delivery Department, Genentech Inc.,
South San Francisco, California 94080, United States
Debby P. Chang −Drug Delivery Department, Genentech Inc.,
South San Francisco, California 94080, United States
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.molpharmaceut.0c00395
Notes The authors declare no competing financial interest.
■ACKNOWLEDGMENTS K.R. acknowledges Fred Lim and Lokesh Kumar for thoughtful scientific discussions and Jasper Lin and Puneet Sharma for carefully reviewing the manuscript.
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Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
Article https://dx.doi.org/10.1021/acs.molpharmaceut.0c00395
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